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Control Strategy for Viral Diseases of Salmonids and Flounder
Yoshimizu, Mamoru
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2003
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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
Yoshimizu
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CONTROL STRATEGY FOR VIRAL DISEASES OF
SALMONIDS AND FLOUNDER
Mamoru Yoshimizu
Laboratory of Microbiology, Graduate School of Fisheries Sciences,
Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
ABSTRACT
Salmonid fish and flounders are economically important fish cultured in northern Japan.
Viral diseases remain one of the limitations to the successful propagation in aquaculture. The
incidence of disease, especially viral disease, has been increasing. Methods currently used to
control viral disease in hatcheries are: (a) hygiene and sanitation, (b) disinfection of water
supplies and wastewater, (c) selection of pathogen- free broodstock, (d) health monitoring of
hatched fry, (e) control of normal intestinal flora, (f) vaccination, and (g) temperature control.
INTRODUCTION
Salmonid fish, such as chum salmon (Oncorhynchus keta), pink salmon (O. gorbuscha),
and masu salmon (O. masou), and flounders, such as Japanese flounder (Paralichthys olivaceus)
and barfin flounder (Verasper moseri), are economically important fish cultured in northern
Japan. Salmonid fish are important species for hatchery reared and released fish. Flounders
are also important fish species for seed production and sea-farming in northern Japan, because
of their rapid growth at low temperatures and relatively high market prices. The incidence of
disease, however, especially viral diseases, has been increasing. Viral diseases found in salmonid
fish are infectious hematopoietic necrosis (IHN), O. masou virus disease (OMVD), erythrocytic inclusion body syndrome (EIBS), chum salmon virus disease, and viral erythrocytic
necrosis. In cultured Japanese flounder and barfin flounder, lymphocystis disease (LCD),
viral ascites, hirame rhabdovirus (HIRRV) disease, viral epithelial hyperplasia, reovirus disease, viral nervous necrosis (VNN), and viral hemorrhagic septicemia (VHS) are found.
Methods currently used to control viral diseases in hatcheries are: (a) hygiene and sanitation, (b) disinfection of water supplies and wastewater using ultraviolet (UV) irradiation, ozonization, and electron decomposition of seawater, (c) selection of pathogen-free broodstock
using antibody detection and virus-specific gene detection by polymerase chain reaction (PCR),
(d) health monitoring of hatched fry by immunological methods and PCR, (e) control of normal intestinal flora (e.g., by feeding an antiviral intestinal bacterium), (f) vaccination (e.g.,
against IHN and OMVD in masu salmon and rainbow trout, O. mykiss, and against LCD in
Japanese flounder) using a formalin-inactivated virus or recombinant G protein expressed via
yeast, and (g) temperature control.
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Control Strategy for Viral Diseases
HYGIENE AND SANITATION
General sanitation measures are standard practice in hatchery and seed producing facilities. Special care must be taken to avoid the movement of equipment from one tank to another
and all articles should be disinfected after use. Methods adopted to sanitize a rearing unit have
to be chosen carefully, due to factors such as chemical toxicity for fish and effects of temperature and repeats use. It should be remembered that workers themselves might serve as efficient vectors for pathogens, and proper disinfection of hands and boots are required to prevent
dissemination of viruses. Although it may be difficult to sanitize a rearing unit during use,
tanks and raceways should be disinfected with a chlorine-based disinfectant before and after
use (Ahne et al. 1989; Kimura and Yoshimizu 1991). Equipment, nets, and brushes are disinfected with ozonated or electrolyzed seawater containing 0.5 mg/L of total residual oxidants
(TROs) or chlorine for 30 min and are used separately for individual tanks (Watanabe and
Yoshimizu 1998; 2001).
Some viruses are transmitted vertically from adult to progeny via infected eggs or sperm.
Disinfection of the surface of fertilized eggs has proven to be effective in breaking the infection cycle for several viruses, such as rhabdovirus, herpesvirus, and nodavirus (Yoshimizu et
al. 1989, Yoshimizu 1998, Watanabe et al. 2000). For IPN, however, surface disinfection may
not always be successful, as the virus may be carried internally in salmonid eggs. The salmonid eggs are disinfected with iodine (50 ppm for 20 min) immediately after fertilization and at
the eyed stage (Yoshimizu and Nomura 1989). Flounder eggs are disinfected with ozonated
seawater (0.5 mg/L of TROs for 10 min) at the morula stage (Watanabe and Yoshimizu 2000).
DISINFECTION OF WATER SUPPLIES AND WASTEWATER
Water systems provide an efficient means for the introduction and spread of infectious
diseases. Pathogen-free water sources are often essential for success in aquaculture. The
surface waters from domestic bay commonly used in aquaculture contain fish pathogens or
animal pathogens. Such open water supplies should not be used without prior treatment to kill
the fish pathogens. Treatment systems usually make use of high efficiency sand filters to
clarify the water before treating it with UV light or ozonization (Kasai et al. 2002).
Fish viruses are divided into two groups, based on their sensitivity to UV and TROs
(Yoshimizu et al. 1986a; 1995; Kasai et al. 2002). Sensitive types are infectious hematopoietic
necrosis virus (IHNV), O. masou virus (OMV), lymphocystis disease virus (LCDV), and Japanese flounder rhabdovirus (HIRRV). These viruses are inactivated by the treatment of 104 ìW
sec/cm2 (UV dose) or 0.1 mg/ml (TROs) for 1 min. Resistant types are infectious pancreatic
necrosis virus (IPNV), marine birnavirus, reovirus, and fish nodavirus, and are inactivated at a
dose of 105 ìW sec/cm2 or 0.5 mg/ml (TROs) for 1 min (Ito et al. 1996a; Kasai and Yoshimizu
2001). Since ozonated seawater that contains TROs shows toxicity to fish, the TROs should
be removed by charcoal (Ito et al. 1996b). Disinfection by electrolysis of seawater for aquaculture and waste seawater from the hatchery is very effective for treating the large volumes of
seawater that are used (Kasai et al. 2000; 2001a; 2001b).
Yoshimizu
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PATHOGEN-FREE BROODSTOCK
Management and monitoring of health conditions are very important for seed production
in aquaculture (Yoshimizu 1998). Health inspections of broodstock are conducted to insure
that fish are free from certain important diseases. Specialized diagnostic techniques are required to make specific pathogen-free broodstock for routine inspections (Yoshimizu and
Nomura 1989). The tests have been made easier and more rapid by the development of enzyme-linked immunosorbent assay (ELISA) (Yoshimizu et al. 1997; 2001; Watanabe et al.
2000).
For salmonid fish, ovarian fluid is collected by the methods of Yoshimizu et al. (1985) and
inspected routinely. Fertilized eggs were disinfected with iodofore 50 ppm for 20 min. Eyed
eggs indicate that inside the egg membrane is pathogen-free (Yoshimizu et al. 1989). Since
viruses and bacteria, including IHNV, OMV, Aeromonas salmonicida, and Renibacterium
salmoninarum, may infect and thrive in the embryo, it is very important to disinfect the surface of eyed eggs with iodofore again.
In flounder hatcheries, tags are applied for identification of individual fish. For example,
to control barfin flounder and Japanese flounder nervous necrosis virus (BFNNV and JFNNV,
respectively), a standard sandwich ELISA uses an expressed protein of partial BFNNV coat
protein for an antigen to capture the specific antibodies, and reverse transcription PCR (RTPCR) to detect striped jack (Caranx vinctus) nervous necrosis virus-specific gene sequences
for broodstock selection. ELISA is done three mo before spawning, and the negative fish by
ELISA are reared for the broodstock. Eggs and sperm are tested by RT-PCR, and specimens
are inoculated to fish cell line SSN-1 cells at the same time. The eggs or sperm that are
positive by RT-PCR are removed (Watanabe et al. 1998; 2000).
HEALTH MONITORING OF HATCHED FRY
Except for Pacific salmon Oncorhynchu, fry from each spawner should be cultured in
separate tanks. When the fry show abnormal swimming or signs of disease, the fish should be
removed and brought to the laboratory for diagnosis as soon as possible. Moreover, health
monitoring should be routinely done, using methods for viral detection such as cell culture,
fluorescent antibody techniques (FAT), immuno-peroxidase stain (IPT), antigen detecting
ELISA, and PCR tests. Reverse transcription PCR (RT-PCR) is suitable for detecting the fish
nodavirus and flounder ascites virus, while FAT is commonly used to diagnose viral epithelial
hyperplasia, lymphocystis disease, HIRRV disease, and reovirus disease (Yoshimizu 1995).
CONTROL OF NORMAL BACTERIAL FLORA
Generally, normal bacterial floras play an important role in inhibiting the growth of pathogenic bacteria in the intestine or on the skin, and in stimulating the immune response of the
host animals. Sometimes, the bacterial flora of larvae cultured in disinfected water is not
normal. It is important to establish the normal bacterial flora of the fish before they are released to the river or ocean.
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Control Strategy for Viral Diseases
Many bacterial strains that produce anti-viral substances against fish viruses have been
reported (Yoshimizu et al. 1986b; Yoshimizu and Ezura 1999). Rainbow trout and masu salmon
that are fed with the bacteria that have been isolated from the normal intestinal flora and which
have an anti-IHNV activity have showed more resistance to artificial infection with IHNV
(Yoshimizu et al. 1992). Vibrio spp. with anti-viral activity against IHNV, OMV and BFNNV
have been isolated from the normal intestinal flora of barfin flounder and added to Artemia
that had been disinfected at the egg stage and hatched out in disinfected water. Anti-IHNV, OMV, and -BFNNV activities have been observed in homogenates of intestines of fish fed
with the Artemia (Yoshimizu and Ezura 1999). Barfin flounder fed with Artemia that contained Vibrio sp. have showed more resistance to natural infection by BFNNV (Yoshimizu and
Ezura 1999).
VACCINATION
Immunization represents the most effective method to control those diseases for which
avoidance is not possible. Commercial vaccines have become available to protect the fish
against important pathogens.
Formalin-inactivated OMV and recombinant IHNV-glycoprotein (IHNV-G protein) expressed by yeast have also been tested. Injecting the OMV vaccine into mature rainbow trout
lowered the incidence of OMV in the ovarian fluid, and an immersion vaccine of IHNV-G
protein expressed yeast has prevented infection by IHNV. Apparently healthy Japanese flounder with low ELISA antibody titers against Japanese flounder lymphocystis disease virus (JFLCDV), have been injected with three types of inactivated JF-LCDV vaccine, including a
formalin-killed Japanese flounder lymphocystis (JF-LC) cell suspension, a heat-killed JF-LC
cell suspension, and a formalin-killed purified JF-LCDV. Three mo post-injection, ELISA
antibody titers had increased to 1:40. In similar fish that had not been injected, the ELISA
antibody titers remained low (1:5 or 1:10). Outbreaks of LCD had been observed at that time.
It was reported that lymphocystis (LC) cells did not grow, but gradually disappeared from
among the immunized fish that showed high ELISA titers. In contrast, it was reported that at
6 mo post-vaccination, LCD was observed among 53 % of the fish that had not been injected
(Yoshimizu 2001; Iwamoto et al. 2001).
TEMPERATURE CONTROL
It is well known that many diseases of aquatic animals are temperature dependent. In the
case of HIRRV, natural outbreaks of disease have disappeared when the water temperature
increases to 15 C. Cumulative mortality of artificially infected Japanese flounder (IP 105.3
TCID50/fish) reared at 5, 10, 15, and 20 C, were reported at 40%, 60%, 10%, and 0%, respectively, with the highest virus infectivity from the fish cultured at 5 C, followed by the 10 C
(Oseko et al. 1988). It has therefore been strongly recommended that Japanese flounder should
be cultured at water temperatures between 15 C and 18 C. No outbreaks of HIRRV disease
have been reported since 1988. Now, temperature control treatment is done against IHN.
Yoshimizu
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